Lens proteins undergo numerous physico-chemical changes during aging and cataract formation; pigmentation and crosslinking are the two major ones. Although several biochemical pathways participate in these changes, the actual mechanisms and their interrelationships remain unclear. Our project addresses this very issue. Reactions of dicarbonyl compounds with proteins appear to be major contributors to pigmentation and crosslinking in lens proteins. Recent studies, including our own, point to yet another mechanism for producing these changes, involving kynurenines, tryptophan oxidation products that react with lens proteins to generate pigments and crosslinking structures. Our preliminary data suggest that the dicarbonyl and kynurenine protein modification processes are related and interdependent. Although most reactions in these pathways are non-enzymatic, the reactive compounds formed can initiate further reactions that are regulated by enzymatic mechanisms. We will investigate these mechanisms in the context of lens physiology. Our proposed research is organized to achieve four specific aims. In the first aim, we will focus on methylglyoxal (MGO), a highly reactive dicarbonyl compound in the lens. We will determine how MGO is metabolized by glyoxalase I as well as the importance of glyoxalase I for survival of lens epithelial cells under conditions of hyperglycemic, oxidative or nitrosative stress. In aim 2, we will establish the role of the dideoxyosone (DDO) pathway in lens protein modification. We will determine how this recently discovered pathway, through which sugars and ascorbic acid modify proteins, contributes to lens aging and cataract formation. In aim 3, we will study kynurenine-mediated protein modification. We will focus mainly on indoleamine 2,3-dioxygenase (IDO), an enzyme that catalyzes metabolism of tryptophan to kynurenines. We will use transgenic and knockout mice to investigate the role of IDO in lens protein modification, and explore the effect of hyperglycemia and oxidative stress on IDO activity. Kynurenine-mediated damage to lens proteins will be assessed by chromatographic methods and immunological assays with highly specific monoclonal antibodies. Finally, in aim 4, we will determine the relationship among kynurenines, MGO and DDO in lens protein modifications. Cummulative information gained from these experiments will provide us with critical insights into the biochemical mechanisms that underlie lens aging and cataract formation.